Fc glycosylation has been an important subject in the field of therapeutic monoclonal antibodies. Fc glycosylation can significantly modify Fc effector functions such as Fc receptor binding and complement activation, and thus affect the in vivo safety and efficacy profiles of therapeutic antibodies.
Several expression systems based on genetically engineering have been reported to produce therapeutic monoclonal antibodies. These include yeasts such as Pichia pastoris, insect cell lines, and even bacteria. However, these expression systems suffer from a number of drawbacks that can negatively affect the effector function of therapeutic antibodies.
The majority of approved biopharmaceuticals are produced in mammalian cell culture systems to deliver proteins with desired glycosylation patterns and thus ensure reduced immunogenicity and higher in vivo efficacy and stability. Non-human mammalian expression systems such as CHO or NS0 cells have the machinery required to add complex, human-type glycans. However, glycans produced in these systems can differ from glycans produced in humans. Their glycosylation machinery often adds undesired carbohydrate determinants which may alter protein folding, induce immunogenicity, and reduce circulatory life span of the drug. Notably, sialic acid as N-acetylneuraminic acid is not efficiently added in most mammalian cells and the 6-linkage is missing in these cells. Engineering cells with the various enzymatic activities required for sialic acid transfer has not yet succeeded in providing a human-like pattern of glycoforms to protein drugs. To date, there is a need for engineering animal cells or glycoproteins to highly sialylated products that resemble as closely as possible to human proteins.
Furthermore, mammalian cell culture delivers a heterogeneous mixture of glycosylation patterns which do not all have the same properties. Properties like safety, efficacy and the serum half-life of therapeutic proteins can be affected by these glycosylation patterns.
Trastuzumab (Herceptin®), approved in 1998 for the treatment of patients with HER2-overexpressing metastatic breast cancers (Baselga et al, (1996) J. Clin. Oncol. 14:737-744), is a humanized anti-HER2 IgG antibody that binds to the extracellular component of the Her2/neu receptor. Overexpression of HER2 is observed in approximately 20% of human breast cancers (hereinafter referred to as HER2-positive breast cancer) and is implicated in the aggressive growth and poor clinical outcomes associated with these tumors (Slamon et al (1987) Science 235: 177-182). Trastuzumab functions with a variety of different of mechanisms, but the main action is to bind to the extracellular membrane portion of the Human Epidermal growth factor Receptor 2 (HER-2) on the surface of cancer cells, preventing the activation of its intra cellular tyrosine kinase. Herceptin acts on the immune system mediating Antibody Dependent Cellular Cytotoxicity (ADCC) and can fix complement, but is considered unable to mediate Complement Dependent Cell Cytotoxicity (CDC).
Trastuzumab is produced in Chinese hamster ovary (CHO) cells and is highly heterogeneous in glycosylation patterns in the Fc domain. Each of anti-HER2 IgG molecules in the heterogeneous mixture may not all have the same property, and certain N-linked oligosaccharides bound to therapeutic proteins may trigger undesired effects in patients thus deeming them a safety concern. Response rates to the antibody Trastuzumab given as a single agent (monotherapy) have ranged from about 15-26%.